Composition comprising a recombinant plasmid and its uses as vaccine and medicament

ABSTRACT

PCT No. PCT/FR95/00345 Sec. 371 Date Sep. 9, 1996 Sec. 102(e) Date Sep. 9, 1996 PCT Filed Mar. 21, 1995 PCT Pub. No. WO95/25542 PCT Pub. Date Sep. 28, 1995A composition containing a recombinant plasmid which includes an exogenous nucleotide sequence capable of expressing a compound including an amino acid sequence in a host organism. The composition includes an emulsion comprising at least one aqueous phase and at least one oily phase, the recombinant plasmid being contained in at least one of phases. A vaccine and a curative drug including the composition are also disclosed.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a new composition comprising arecombinant plasmid, as well as to vaccines or curative medicamentscomprising such a composition.

2. Description of the Related Art

Vaccination with live viruses or microorganisms has numerous well-knownadvantages compared to vaccination with non-live vaccines consisting ofkilled microorganisms or of isolated proteins or peptides.

Live vaccines consist of a live microorganism or a live virus. Theselatter are generally attenuated so as to decrease the pathogenic risks.However, this risk is never completely eliminated inasmuch as, inparticular, a reversion of the microorganism or virus to a virulent formmay occur.

Moreover, the mutations or deletions employed in order to bring about anattenuation of the virulence of the strains sometimes cause a decreasein the immune response, necessitating the injection of large doses ofvaccines. Recently, new vaccines whose preparation has been madepossible by the advances in genetic engineering have been developed.Among these new vaccines, there may be mentioned those consisting of arecombinant plasmid formed from a nucleotide sequence into which isinserted an exogenous nucleotide sequence originating from amicroorganism or from a pathogenic virus. The purpose of the latternucleotide sequence is to permit the expression of a compound comprisingan amino acid sequence, the purpose of this compound itself being totrigger an immune reaction in a host body.

The first injection of such a plasmid, as well as its expression in amuscle, was performed and demonstrated in 1990 by Lin et al.(Circulation 82:2217-2221). The object of this experiment was todemonstrate that a recombinant plasmid injected into a living organismwas capable of expressing the exogenous sequence in the tissues intowhich had been injected. It was demonstrated in this way that theadministration of a recombinant plasmid to a living organism could beused in gene therapy. This therapeutic method is well known andconsists, in particular, in administering to a host body geneticallymodified cells or, as demonstrated by Lin et al., recombinant plasmidscapable of expressing compounds synthesized by living organisms, such aspeptides, proteins or glycoproteins. The purpose of the in vivosynthesis of these compounds can be either to compensate for adeficiency of a genetic nature in the host body to which the geneticallymodified cells of the recombinant plasmids have been administered, or tohave a curative action against a disease, such as a cancer, triggered inthis same host body.

As an example, such a curative action may consist of a synthesis, byrecombinant cells or recombinant plasmids, of cytokines such asinterleukins, in particular interleukin-2. These cytokines enable animmune reaction directed towards the selective elimination of cancercells to be triggered or enhanced.

Subsequently, it was shown that recombinant plasmids carrying a specificgene, coding for the glycoprotein of bovine herpesvirus I (BHV-1), werecapable of inducing an immune response, consisting of the synthesis ofantibody, in different animal species, namely mice and cattle (Cox etal., J. Virol. September 1993, 67, 9, 5664, 5667). However, this studyalso shows clearly a heterogeneity of the immune responses obtained. Inother words, all other conditions being equal, the immune response ishighly variable for each animal tested and each injection performed. Itemerges from this that a few animals display a low level of antibodies,though sometimes sufficient to induce some degree of protection, whereasmost of the other animals do not display an antibody level of this kind.In the latter case, no protection is induced.

SUMMARY OF THE INVENTION

The present invention thus consists of a composition comprising arecombinant plasmid capable of expressing, in a host body to which saidrecombinant plasmid has been administered, a larger amount of compoundsof the peptide, protein or glycoprotein type.

Another subject of the invention consists of a composition containing arecombinant plasmid at a concentration lower than that in traditionalcompositions, with a view to an expression of compounds of the peptide,protein or glycoprotein type in an unchanged or even a larger amount.

Another subject of the invention consists of a vaccine comprising acomposition containing a recombinant plasmid enabling a more homogeneousimmune response and an enhanced protection against a virus or apathogenic microorganism to be obtained.

Yet another subject of the invention consists of a curative medicamentcomprising a composition containing a recombinant plasmid capable ofexpressing increased amounts of molecules of the peptide, protein orglycoprotein type, and of being employed with a reduced concentration ofrecombinant plasmids.

Thus the invention consists of a composition containing a recombinantplasmid comprising an exogenous nucleotide sequence capable ofexpressing a compound comprising an amino acid sequence in a host body,characterized in that said composition comprises an emulsion containingat least one aqueous phase and at least one oil phase, said recombinantplasmid being contained in at least one of said phases.

BRIEF DESCRIPTION OF THE DRAWING

The FIGURE shows the results of a challenge resistance test performed onmice to which either a recombinant plasmid carrying the GP50 gene of theAujeszky virus or various controls was/were administered.

DESCRIPTION OF THE PREFERRED EMBODIMENT

A recombinant plasmid according to the invention may be preparedaccording to standard methods, for example the method described in thepaper by Cox et al., J. Virol, September 1993, 67, 9, 5664-5667,mentioned above, or in the paper by M. Eloit et al., J. of General Virol(1990), 71, 2425-2431 (especially FIG. 1). The teaching of the latterpaper is incorporated herein for reference. Naturally, the exogenousnucleotide sequence varies in accordance with the compound to beexpressed.

The compound capable of being expressed by the recombinant plasmid whenit has been administered to the host body can be a peptide, a protein ora glycoprotein.

This compound can consist, in particular, of an antigen capable oftriggering in said host body an immune reaction with respect to apathogenic virus or to a pathogenic microorganism. This immune reactionmay be of the humoral or cellular type, and may consist, in particular,of a synthesis of antibody enabling a protection with respect to such avirus or such a microorganism to be conferred on the host body. By wayof such pathogenic viruses, the Aujesky virus, an HIV virus such asHIV-I or HIV-II, an FIV virus or a flu virus of the influenzae type maybe mentioned in particular. In context of the present invention,microorganism is understood to mean a bacterium, a yeast, a fungus, amycoplasma or a unicellular parasite. By way of pathogenic bacteria,furunculoses sic!, Escherichia coli or bacteria of the generaPastorella, Salmonella or Yersinia may be mentioned in particular. Byway of pathogenic yeasts, those of the genus Candida, such as Candidaalbicans, may be mentioned. By way of unicellular parasites, Plasmodiumfalciparum or parasites of the genus Leishmania may be mentioned.

Usually, the exogenous nucleotide sequence originates from such apathogenic virus or microorganism.

Alternatively, the recombinant plasmid administered to the recipienthost can comprise a nucleotide sequence capable of expressing a compoundcomprising an amino acid sequence, such as a peptide, a protein or aglycoprotein, having a curative action with respect to a disease,especially a noninfectious disease possibly of a functional nature whichhas been triggered in the host body. In this case, the recombinantplasmid permits a therapeutic treatment of the gene therapy type asdefined above. Thus, the function of said compound may be to compensatefor a deficiency of a genetic nature in the host body, for the purposeof treating a genetic disease such as cystic fibrosis or myopathy. Saidcompound may also have a curative action with respect to a functionaldisease which has become manifest in the host body; for example, it mayhave a curative action with respect to cancer cells. This curativeaction may consist of the synthesis of cytokines, for instanceinterleukins such as interleukin-2.

The host body in which said recombinant plasmid must be capable ofexpressing a compound comprising an amino acid sequence may be ananimal, or even a tissue of an animal, such as an insect or a vertebrateanimal, especially a mammal, a fish or a bird. Such a mammal may be man,a canine, a bovine, a pig, a rabbit, an ovine or a feline. By way of abird, gallinaceans such as chickens may be mentioned.

A composition according to the invention can contain an oil-in-water(O/W), water-in-oil (W/O) or water-in-oil-in-water (W/O/W) typeemulsion. An especially preferred type of emulsion in the context of theinvention consists of an O/W emulsion. An emulsion according to theinvention may be prepared according to traditional methods ofpreparation of an emulsion, in particular according to the processesdescribed in Patent Applications EP-A-489,181 and EP-A-481,982. Thus,the oil constituting the oil phase may be emulsified while stirring itwith the aqueous phase consisting of an aqueous solution or suspensioncontaining the recombinant plasmid. Alternatively, the composition ofthe invention may be prepared by emulsifying an oil comprising therecombinant plasmid, for example in lyophilized form, with an aqueousphase.

An emulsion according to the invention can contain, by weight, from 5 to95% of oil phase for 95 to 5% of aqueous phase, and preferably from 25to 75% of oil phase for 75 to 25% of aqueous phase. The emulsion must bestable, preferably for at least 12 months when it is stored at 4° C.

The oil constituting the oil phase can be a mineral oil, a nonmineraloil or a mixture of a mineral oil and a nonmineral oil. Said mineraloils may be natural or synthetic. Said nonmineral oils may be ofvegetable, animal or synthetic origin. All these oils lack toxic effectswith respect to the host body to which the composition of the inventionis administered. They are preferably liquid at the storage temperature(approximately +4° C.), or at least to make it possible sic! to giveliquid emulsions at this temperature. An advantageous mineral oilaccording to the invention can consist of an oil comprising a linearcarbon chain having a number of carbon atoms which is preferably greaterthan 16, and free from aromatic compounds. Such oils can be, forexample, those marketed under the name "MARCOL 52" (produced by EssoFrance) or "DRAKEOL 6VR" (produced by Penreco USA), which are bothcommercial mineral oils having a linear hydrocarbon chain, free ofaromatic compounds.

By way of synthetic organic oils, polyisobutenes or polyisopropenes maybe mentioned. Among vegetable oils, oleic acid-rich unsaturated oilswhich are biodegradable may be mentioned, for example groundnut, olive,sesame, soybean or wheat germ oils.

The animal oils can consist, in particular, of squalene, squalane orspermaceti oil.

Besides the oil phase and the aqueous phase, the composition accordingto the invention, in particular when it is used as vaccine, may containan immune stimulating agent such as avridine.

Moreover, the composition according to the invention may alsoadvantageously contain a surfactant. The latter displays a lipophilic orhydrophilic character characterized by an HLB (hydrophilic-lipophilicbalance) value of between 1 and 19.

A preferred surfactant in the context of the present invention canconsist of an ester obtained by condensing a fatty acid, advantageouslya fatty acid which is liquid at 20° C., with a sugar or glycerol. Saidsugar can consist of glucose, sucrose or, preferably, mannitol. By wayof an especially preferred mannitol ester, there may be mentionedmannitol oleates obtained by anhydridizing the polyhydroxylated carbonchain of mannitol which is cyclized at positions 1-4 or 2-6.

Derivatives of these esters may also be employed. These derivativesdisplay a hydrophilicity which is modified, in particular, by graftingof hydrophilic functions such as alcohol, polyol, ethylene oxide,propylene oxide, carboxylic acid, amine or amide. A surfactant accordingto the invention is preferably pharmaceutically acceptable for use as aninjectable preparation; it must, in particular, lack heavy metals andpossess very low acid or peroxide values. It is also desirable for it tosatisfy the specifications of a safety test such as, for example, theone described by S. S. Berlin, Annales of Allergy, 1962, 20, 473.Preferably, the surfactant is combined with the oil before formation ofthe emulsion.

Oils combined with a surfactant which are most especially suitable inthe context of the present invention are those marketed by the companySEPPIC under the brand name "MONTANIDE" (mixture of oil and surfactant).The properties of these oils appear in Table 1 below.

                                      TABLE 1                                     __________________________________________________________________________                         Aqueous                                                                       phase/    Conduc-                                                             emulsion  tivity                                                         Emulsion                                                                           (% by                                                                              Viscosity                                                                          at 25° C.                               No.                                                                              Trade name                                                                           Oil   type weight)                                                                            (mPa · s)                                                                 (μS · cm.sup.-1)                   __________________________________________________________________________    1  MONTANIDE                                                                            Mineral                                                                             O/W  75%  20   5000                                              ISA 25                                                                     2  MONTANIDE                                                                            Mineral +                                                                           O/W  75%  20   5000                                              ISA 25A                                                                              avridine*                                                           3  MONTANIDE                                                                            Mineral +                                                                           O/W  75%  25   1000                                              ISA 28 nonmineral                                                          4  MONTANIDE                                                                            Mineral                                                                             W/O/W                                                                              50%  50   1000                                              ISA 206                                                                    5  MONTANIDE                                                                            Mineral                                                                             W/O  50%  200  1                                                 ISA 50                                                                     6  MONTANIDE                                                                            Nonmineral                                                                          W/O  30%  70   1                                                 ISA 708                                                                    __________________________________________________________________________     *Avridine = N,NDioctadecyl-N-bis(2-hydroxy-ethyl)propanediamine.         

A composition according to the invention possesses a viscosity of lessthan 300 mPa.s, and advantageously less than 200 mPa.s, measured at 25°C. by means of a Brookfield type rotational viscometer. The "oily" or"aqueous" character of the continuous phase of the emulsion ischaracterized by the conductivity measured in microsiemens per cm(μS.cm⁻¹) at 25° C. Values below 20 μS.cm⁻¹ indicate the presence of acontinuous oil phase.

A composition according to the invention comprises an amount ofrecombinant plasmid which is variable in accordance, mainly, with thehost body and the nature of the compound expressed by said plasmid.

Usually, a composition according to the invention can comprise from 0.01to 100 g/l of said recombinant plasmid.

According to another aspect, the invention also relates to a vaccinecomprising a composition as defined above. The purpose of such a vaccineis to provoke an immune reaction against a virus or a pathogenicmicroorganism such as the ones mentioned above.

According to yet another aspect, the invention relates to a curativemedicament comprising a composition as defined below. The purpose ofsuch a medicament is to treat a disease, in particular a functionaldisease of the host body to which it is administered.

These vaccines and curative medicaments comprising said composition aregenerally in an injectable form. The host body to which they can beadministered are the animals or even animal tissues mentioned above.Man, dogs, cats, poultry, oxen, sheep, pigs and horses will be mentionedhere more especially.

The dose of recombinant plasmid contained in said composition to beadministered depends on the nature of the host body. This dose ofrecombinant plasmid can thus vary very widely between 1 μg and 500 mg/kghost body weight.

As an example, it is possible to administer a dose of recombinantplasmid of 500 mg/kg to a mouse weighing 20 g, of 1 μg/kg to an oxweighing 500 kg and a dose of 100 μg to 100 mg/kg, and preferably 1 mgto 30 mg/kg, in an adult human.

A composition according to the invention may be used more especially toprepare a vaccine intended to produce an immune reaction in a host body,for prevention against an HIV virus (in particular in man), an Aujeszkyvirus (in particular in pigs) or an FIV virus (in particular in cats).

A composition according to the invention may also be used to prepare amedicament intended to treat a cancer.

The purpose of the examples which follow is to illustrate the presentinvention.

All these examples employ a recombinant plasmid carrying the gp50 geneof the Aujeszky virus. This was obtained according to the methoddescribed in the paper by M. Eloit et al., J. of General Virol (1990),71, 2425-2431 (especially FIG. 1) mentioned above.

In these examples, the following terms mean:

pGP50-ISA 25 (according to the invention): a vaccine containing saidrecombinant plasmid carrying the GP50 gene of the Aujeszky virus in anemulsion containing MONTANIDE ISA 25 as oil phase (see Table 1);

pGP50+ISA 25 (not according to the invention): a vaccine injected in twostages: in a first stage, an aqueous suspension containing saidrecombinant plasmid carrying the GP50 gene of the Aujeszky virus isinjected, and in a second stage, an emulsion (not containing anyplasmid) containing MONTANIDE ISA is injected;

pGP50 (not according to the invention): a vaccine consisting only of anaqueous phase comprising the recombinant plasmid;

pSU-ISA 25 (control): a vaccine similar to pGP50-ISA 25, but comprisinga control plasmid expressing an exogenous protein pSU, different frompGP50;

pSU+ISA 25 (control): a vaccine similar to pGP50-ISA 25, but comprisinga control plasmid expressing pSU instead of pGP50;

pSU: a vaccine similar to pGP50, but comprising a control plasmidexpressing pSU instead of pGP50.

EXAMPLE 1

Mice numbered 1 to 6 are injected with different vaccines.

At regular intervals, the antibodies produced by these vaccines areassayed. For this purpose, the serum is drawn from the mice and theanti-GP50 antibodies are assayed by an ELISA technique described by M.Eloit et al. in Veterinary Record, 1989, 124, 91-94. The results appearin Tables 2 to 5 below. They are expressed in terms of the last dilutionnecessary for obtaining an optical density above the background.

The results obtained show that a vaccine according to the inventionmakes it possible to obtain an antibody level markedly higher than thatobtained with the comparative vaccines or the controls, as well as agreater homogeneity of the immune response. This homogeneity manifestsitself in the obtaining of a high level of antibody, enabling aneffective protection to be induced in a larger number of animals.

                  TABLE 2                                                         ______________________________________                                        Titration of anti-GP50 antibodies by                                          ELISA assay 2 weeks after vaccination:                                                 mouse   mouse   mouse mouse mouse mouse                              Vaccine  1       2       3     4     5     6                                  ______________________________________                                        pGP50 - ISA                                                                            32      1024    32    1024  --    --                                 25                                                                            pGP50 + ISA                                                                            16      16      32    16    --    --                                 25                                                                            PGP50    16      8       8     16    256   16                                 pSU - ISA                                                                              <8      <8      <8    <8    --    --                                 25                                                                            pSU + ISA                                                                              <8      <8      <8    <8    --    --                                 25                                                                            pSU      <8      <8      <8    <8    --    --                                 ______________________________________                                    

                  TABLE 3                                                         ______________________________________                                        Titration of anti-GP50 antibodies by                                          ELISA assay 4 weeks after vaccination:                                                 mouse   mouse   mouse mouse mouse mouse                              Vaccine  1       2       3     4     5     6                                  ______________________________________                                        pGP50 - ISA                                                                            32      384     32    1024  --    --                                 25                                                                            pGP50 + ISA                                                                            16      8       8     8     --    --                                 25                                                                            pGP50    16      8       8     16    128   8                                  pSU - ISA                                                                              <8      <8      <8    <8    --    --                                 25                                                                            pSU + ISA                                                                              <8      <8      <8    <8    --    --                                 25                                                                            pSU      <8      <8      <8    <8    --    --                                 ______________________________________                                    

                  TABLE 4                                                         ______________________________________                                        Titration of anti-GP50 antibodies by                                          ELISA assay 6 weeks after vaccination:                                                 mouse   mouse   mouse mouse mouse mouse                              Vaccine  1       2       3     4     5     6                                  ______________________________________                                        pGP50 - ISA                                                                            1536    2048    --    2048  --    --                                 25                                                                            pGP50 + ISA                                                                            128     128     128   48    --    --                                 25                                                                            pGP50    384     64      256   512   128   128                                pSU - ISA                                                                              <8      <8      <8    <8    --    --                                 25                                                                            pSU + ISA                                                                              <8      <8      <8    <8    --    --                                 25                                                                            pSU      <8      <8      <8    <8    --    --                                 ______________________________________                                    

                  TABLE 5                                                         ______________________________________                                        Titration of anti-GP50 antibodies by                                          ELISA assay 3 months after vaccination:                                                mouse   mouse   mouse mouse mouse mouse                              Vaccine  1       2       3     4     5     6                                  ______________________________________                                        pGP50 - ISA                                                                            2048    1280    --    768   --    --                                 25                                                                            pGP50 + ISA                                                                            256     256     256   256   --    --                                 25                                                                            pGP50    384     128     256   256   192   256                                pSU - ISA                                                                              <8      <8      <8    <8    --    --                                 25                                                                            pSU + ISA                                                                              <8      <8      <8    <8    --    --                                 25                                                                            pSU      <8      <8      <8    <8    --    --                                 ______________________________________                                    

EXAMPLE 2

6 groups of 6 mice each are vaccinated with a different vaccine:

pGP50,

pGP50-ISA 25,

pGP50+ISA 25,

pSU-ISA 25,

PSU+ISA 25, and

pSU.

Six weeks after injection of the vaccine, each mouse is injected with anaqueous suspension containing a virulent Aujeszky virus.

The percentage survival of the mice after this latter injection is thendetermined.

The results obtained are presented in the figure, where the survivalrate is plotted as ordinate. These results show that a vaccine accordingto the invention, pGP50-ISA 25, enables twice as large a protection tobe conferred as with a vaccine of the prior art comprising only a pGP50recombinant plasmid in aqueous suspension.

What is claimed is:
 1. A composition containing:(a) a recombinantplasmid comprising a nucleotide sequence encoding a protein,glycoprotein, or peptide; and (b) an emulsion comprising one or twoaqueous phases and an oil phase containing a surfactant having ahydrophilic-lipophilic balance (HLB) value of between 1 and 19, saidrecombinant plasmid being contained in at least one of said phases. 2.The composition according to claim 1, wherein said recombinant plasmidencodes a protein, glycoprotein, or peptide of Aujeszky virus.
 3. Thecomposition according to claim 1, wherein said recombinant plasmidencodes the gp50 glycoprotein of Aujeszky virus.
 4. The compositionaccording to claim 1, wherein said emulsion is oil-in-water (O/W),water-in-oil (W/O) or water-in-oil-in-water (W/O/W).
 5. The compositionaccording to claim 4, wherein said emulsion is O/W.
 6. The compositionaccording to claim 1, wherein said surfactant is an ester obtained bycondensing a fatty acid with mannitol.
 7. The composition according toclaim 1, having a viscosity of less than 300 mPa.s at 25° C.